1. Field of the Invention
The present invention relates generally to the field of molecular biology. More specifically, the invention relates to methods and compositions for increasing plant seed yield.
2. Description of the Related Art
BRS1 is a secreted serine carboxypeptidase that is implicated in an early step in brassinosteroid signaling, probably by taking part in the proteolytic processing of a protein involved in activating the BRI1 receptor (Li et al., 2001). The protease activity of BRS1 is required for its function in suppressing the phenotypes of a weak BRI1 allele, bri1-5. BRI1 is a member of a serine carboxypeptidase gene family in Arabidopsis. The fact that a loss-of-function allele of BRS1 does not show any significant phenotypes suggests that there is functional redundancy among the family members.
It has been shown that BRS1 overexpression suppresses multiple bri1 defects, suggesting BRS1 might play an important role in an early stage of the BRI1 signaling pathway (Li et al., 2001). The presence of an N-terminal signal peptide in BRS1 predicts that the protein should enter the secretory pathway. Sequence analysis failed to identify any obvious endoplasmic reticulum or Golgi apparatus retention sequences. Therefore, BRS1 may be a secreted protein. These observations are consistent with findings that BRS1 suppressed two extracellular domain mutants, bri1-5 and bri1-9, but failed to suppress a loss-of-function cytoplasmic domain mutant bri1-1 (Friedrichsen et al., 2000).
BRS1 shares homology with another serine carboxypeptidase II-like protein, designated ECS1. Like BRS1, ECS1 is predicted to have an N-terminal signal peptide and should be secreted. Based on its biochemical properties, yeast Kex1p is classified in the same carboxypeptidase group (carboxypeptidase D). In yeast, both Kex1p and Kex2p/kexin are required for the maturation of peptide hormones, α-mating pheromone and K1 killer toxin, from their inactive precursors (Dmochowska et al., 1987; Fuller, 1989). Kex2p/kexin is a membrane bound endoprotease, which specifically cleaves on the carboxyl side of pairs of basic amino acids (e.g. KR↓ or RR↓). Kex2p related endoproteases are also known as subtilisin and furin (Barr, 1991). Following the action of Kex2p/Kexin, Kex1p selectively trims off the flanking amino acids from the C-terminus of processing intermediates.
There are numerous examples of the importance of carboxypeptidases in ligand processing in animals. For example, a mutation in carboxypeptidase E (CPE), a metallopeptidase, results in the fat mouse mutant (Naggert et al., 1995; Fricker and Leiter, 1999). CPE is widely distributed in brain, pituitary and other neuroendocrine tissues and is thought to be involved in the processing the precursors of neuroendocrine peptides (Naggert et al., 1995; Fricker and Leiter, 1999).
In addition to ligand processing, there are also examples of receptor proteolytic processing. One example of receptor processing is the insulin receptor. Both insulin and insulin receptor are synthesized as inactive precursors. Proinsulin and insulin proreceptors are processed by furin-like endoproteases in the trans Golgi network to form active molecules, which recognize and cleave at the carboxy terminal sites of dibasic amino acids. Proinsulin is processed at the C-termini of KR and KTRR sites. The insulin proreceptor is processed at the RKRR site (Barr, 19991).
In plants, there are a few reports concerning the processing of ligand-like peptides or receptor-like proteins. In response to wounding, tomato systemin is processed from its inactive form, preprosystemin (Schaller and Ryan, 1994). Also in tomato, a secreted leucine-rich repeat protein (LRP), which was thought to be involved in a plant defense response, is proteolytically processed during pathogenesis (Tornero et al., 1996). It is not clear whether prosystemin is cleaved by a subtilisin-like endoprotease, but it has been found that systemin physically interacts with a subtilisin-like protein SPB50 (Schaller and Ryan, 1994). LRP is likely to be processed by a subtilisin/Kex2p-like endoprotease (Tornero et al., 1996). Additionally, the functions of two Arabidopsis Kex2p-like genes have been determined: AIR3 is involved in the regulation of auxin-induced lateral root formation (Neuteboom et al., 1999) and SDD1 functions in guard cell development (Berger and Altmann, 2000).
The regulatory roles of serine carboxypeptidases in plants have not yet been investigated. Therefore, while the foregoing studies have further understanding of plant metabolism, a beneficial use for numerous serine carboxypeptidases and for ECS1 and its orthologs in particular has been lacking.